The invention relates to the use of lactoferrin as an agent for the propylactic and therapeutic treatment of the toxic effects of endotoxins.
Lactoferrin is a glycoprotein with an average molecular weight of 77,000 Dalton. It can bind two molecules of iron reversibly. It can also bind other bivalent or trivalent metal ions instead of iron such as copper, magnesium, zinc, manganese and cobalt.
Lactoferrin was first isolated from milk in 1960. The lactoferrin content of human milk is approx. 1 mg/dl. Lactoferrin has also been found in the blood, in the granula of the neutrophil granulocytes, in lacrimal fluid, in gastric fluid and in the secretions of the intestinal tract. The lactoferrin concentration found in the blood ranges from 40 to 200 .mu.g/dl.
Lactoferrin in the blood, which is released mainly by the granula of the neutrophil granulocytes, contributes to some aspects of iron transport to the cells of the reticulo-endothelial system, especially during infections or inflammatory processes. [Van Snick JL, Masson L, Heremans JF. The involvement of lactoferrin in the hyposideremia of acute inflamation. J Exp Med 1974; 140: 1068-1084]. In animal experiments, due to the rapid release of lactoferrin from the granulocytes, the administration of endotoxin or bacteria was seen to be nearly concurrent with the increase in plasma lactoferrin levels. For this reason, an increase in plasma lactoferrin concentration is considered to be an early indicator for endotoxemia or septicemia (Gutteberg TJ., Rokke O., Joergensen T.; Andersen O.,: Lactoferrin as an Indicator of septicemia and Endotoxemia in Pigs. Scand J Infect Dis (1988), 20: 659-666).
Another essential function of lactoferrin, particularly in the neonatal period, is the regulation of iron absorption in the intestinal tract [Brock JH, Lactoferrin in human milk: its role in iron absorption and protection against enteric infections in the newborn infant. Arch Dis Child (1980) 55: 417-422], since the lactoferrin in human milk is capable of releasing the bound iron to the cells of the gut mucosa.
The most important biological function of lactoferrin derives, however, from its bacteriostatic effect [Arnold RR, Brewer M, Gauthier JJ. Bactericidal activity of human Lactoferrin: Sensitivity of a variety of microorganisms. Infection and Immunity (1980) 28: 893-898; Arnold RR, Russel JE, Champion WJ, Gauthier JJ. Bactericidal activity of human Lactoferrin: influence of physical conditions and metabolic state of the target microorganisms. Infection and Immunity (1981) 32: 655-660; Gutteberg T.J., Rokke O., Andersen O., Joergensen T.; Early Fall of Circulating Iron and Rapid Rise of Lactoferrin in Septicemia and Endotoxemia: An Early Defence Mechanism. Scand J Infect Dis,(1989) 21: 709-715]. Due to its bacteriostatic effect, the lactoferrin in human milk plays an important role as a defense mechanism in that it protects the newborn against intestinal infections. This bacteriostatic effect results from the high iron-binding capacity of lactoferrin. Iron is an essential growth factor for bacteria. The complexing of iron and lactoferrin keeps the concentration of free iron around the bacteria below the level conducive to bacterial growth. This results in an inhibition of bacterial growth. The higher the iron content, the less bacteriostatic effect lactoferrin has. Since iron-free lactoferrin (apoform) is released as early as the initial phase of bacteriemia and endotoxemia, GUTTEBERG et al. assumed that iron-free lactoferrin, in view of its bacteriostatic effect, might be an early defense mechanism against bacterial invasion [Gutteberg T.J., Rokke O., Andersen O., Joergensen T.; Early Fall of Circulating Iron and Rapid Rise of Lactoferrin in Septicemia and Endotoxemia: An Early Defence Mechanism. Scand. J. Infect. Dis.,(1989), 21: 709-715]. The publication cited above does not contain any evidence that the iron-free apoform of lactoferrin released from granulocytes plays the role of a defense mechanism against the endotoxin in addition to its function as a defense mechanism against bacteria.
Lactoferrin also seems to fulfil some function in the physiological regulation of granulopoiesis, whereby the mechanism involved is not yet fully understood. It has been observed that the release of the colony stimulating factor (CSF) from the macrophages can be inhibited by lactoferrin. This factor is essential for granulopoiesis. The inhibition of CSF release depends on the iron load of the lactorferrin [Broxmeyer HE et al. Identification of lactoferrin as the granulocyte-derived inhibitor of colony-stimulating activity production. J Exp Med 1978; 148: 1052-1068].
Endotoxin is a constituent of the cellular walls of gram-negative bacteria and is released only by the bacterial decay. It is a macromolecule with a molecular weight of up to 1.times.10.sup.6 Dalton, consisting mainly of sugar compounds and fatty acids. It may also include complexed protein residues from the wall of the bacteria. The endotoxin molecule consists of three structurally and immunobiologically different subregions:
Subregion 1, the O-specific chain, consists of several repetitive oligosaccharide units, each of which is made up of a maximum of 5 neutral sugars. The number of oligosaccharides present depends on the strain of bacteria; for example, the endotoxin of S. abortus equi used in our experiments has 8 oligosaccharides in this region.
Subregion 2, the core oligosaccharide, consists, among other things, of n-acetyl-glucosamine, glucose, galactose, heptose and 2-keto-3-desoxyoctone acid.
Subregion 3, The lipid A (MW 2,000 Dalton) consists of a phosphorylated D-glucosamine-disaccharide to which several - approx. 7 - long-chain fatty acids are bound as amides and esters. The carrier of the toxic properties is the lipid A, whereby the toxic effects derive from several fatty acid residues in this region.
The size of the endotoxin molecule and its charge characteristics allow for complexing various compounds and proteins with the groups or side-chains of the three subregions in the endotoxin structure without this having any influence on its toxic properties. Normally, there is a protein bound to the lipid A, the so-called lipid A-associated protein. In most cases, separation of this protein component from the endotoxin causes no change whatever in the toxic effect. It was, however, found that binding of proteins onto many endotoxins can also result in a considerable increase in their toxicity (Rietschel E.TH. et al. (1982)): "Bacterial Endotoxins: Chemical Structure, Biological Activity and Role in Septicaemia", Scand. J. Infect. Dis. Suppl. 31: 8-21; p. 17).
There is also a physiological transfer of endotoxin from the intestine into the blood in healthy persons. Elimination of endotoxin from portal vein blood seems to occur mainly in the liver. To protect organs from damage, plasma in healthy persons can inactivate the endotoxin that is continually transferred from the intestine, although the mechanism has not yet been explained. Intestinal permeability disturbances may result in increased transfer of endotoxin from the intestine into the bloodstream. Such disturbances occur, for example, following disturbances of microcirculation due to shock or inflammatory intestinal diseases or under immunosuppressive therapy with Ciclosporin A. Increased endotoxin transfer from the intestine into the blood is also observed following enteral antibiotic therapy leading to increased endotoxin release in the intestine (NITSCHE D, STEHLE M, HAMELMANN H, Der Einflu.beta. der Immunsuppression mit Ciclosporin auf die enterogene Endotoxinamie: Tierexperimentelle Untersuchungen. Langenbecks Archiv fur Chirurgie, 1990, Suppl.). Release of large amounts of endotoxins, and therefore increased transfer of endotoxin from the septic focus into the bloodstream may also occur in cases of extensive gram-negative infection such as peritonitis. Such a massive influx of endotoxins exhausts the plasma's capacity to inactivate these endotoxins quickly enough, leading to higher plasma levels of biologically active endotoxin and resulting, finally in clinical endotoxemia. On the one hand, endotoxin causes the release of toxic mediators; on the other hand, it also disturbs the energy metabolism of the cell, resulting in cell decay and--in the case of a higher dosis of endotoxin and/or a longer duration of the endotoxemia - organ failure as well. For this reason, all diseases involving the possibility of increased endotoxin transfer from the intestine or from a septic focus into the blood, or which involve a disturbance of the physiological elimination of endotoxin by the liver, require additional therapeutic measures capable of reducing endotoxin activity in the blood.
An important additional therapeutic measure in treatment of endotoxemia is the reduction of the influx of endotoxins from the intestine, which is also continuous under physiological conditions.
Oral administration of substances capable of adsorbing endotoxin has been proposed as a therapeutic measure to reduce the influx of endotoxin from the intestine into the blood [Ditter B, Urbaschek R, Urbaschek B. Ability of various adsorbents to bind endotoxins in vitro and to prevent orally induced endotoxemia in mice. Gastro-enterology 1983; 84:1547-1552]. The main problem involved in using these substances is that they succeed in binding the endotoxins in the intestine, but do not inactivate their toxic properties, so that toxic endotoxins might be released once again as a result of a chemical change in the ambient milieu.
As demonstrated by clinical trials, it is possible to reduce plasma endotoxin activity by administering monoclonal lipid A antibodies [Ziegler EJ et al; Treatment of gram-negative bacteremia and septic shock with HA-1A human monoclonal antibody against endotoxin. N Eng J Med, 1991:429-436]. Plasma endotoxin activity can also be reduced to a certain degree by administering polyvalent immunoglobulin preparations, since they can be expected to contain a certain amount of lipid A antibodies. With the exception of the preparations containing monoclonal lipid A antibodies, however, all of the therapeutic means available to date are marred by the disadvantage that they inactivate only a certain percentage of the endotoxins entering the blood. For this reason, they prove ineffective when larger amounts of endotoxins enter the bloodstream.